This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Atmospheric nitrogen fixation is a tremendous chemical feat accomplished by a few select microorganisms possessing nitrogenase, a unique metalloenzyme. Nitrogenase derives its competency from several iron-sulfur clusters that form a chain of electron transport. Two of these metal clusters, the P-cluster and FeMoco, possess an exceptional structure and composition that make them exciting subjects of study. Little is understood about the biosynthesis of these two complex metalloclusters. Progress made in unraveling the mechanism of their assembly would be invaluable in better understanding not only nitrogen fixation, but also biological metal cluster assembly as a whole. A series of x-ray absorption studies are proposed to ascertain the specific stepwise process through which these clusters are constructed. The first examination will focus on the biosynthesis of P-cluster. In a previous work, a non-native conformation of the Pcluster on a FeMoco-free nitrogenase MoFe protein (designated Av1) was identified. Pre-incubation of this form of MoFe protein at various time lengths with the nifH gene product, the nitrogenase Fe protein (designated Av2), will be performed prior to quench. Determination of the changes in the average iron coordination, oxidation state, and atomic environment by Fe K-edge XAS/EXAFS would render a time-lapse capture of P-cluster assembly and suggest a role of the Fe protein in P-cluster maturation. This will be coupled to concomitant EPR and activity assay analysis, providing a complete picture of P-cluster generation. The second examination will focus on the assembly of FeMoco. A unique assay has been developed that permits a previously unseen transformation of a significant fraction of the iron-only precursor into the mature FeMoco on the NifEN scaffold protein. Determination of the structure of this novel reconstituted precursor by Mo K-edge XAS/EXAFS may identify intermediates in FeMoco construction and establish structural and conditional requisites for FeMoco reactivity.